Constitutive activation of a nuclear-localized calcium channel complex in Medicago truncatula

Nuclear Ca²⁺ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca²⁺ oscillations. However, the mechanism of Ca²⁺ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca²⁺ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca²⁺ signals, and provide insights into its native mode of induction.